This invention concerns peptides. More particularly, the invention concerns compositions for administration to dogs, which actively provide immunity to the dog""s immunoglobulin E molecules.
It is estimated that up to 30% of all dogs suffer from allergies or allergy-related skin disorders. Specifically, allergic dermatitis has been estimated to affect between 3 and 15% of the entire canine population. Given the prevalence of allergies in dogs, there is a need to develop methods and compositions to properly diagnose and treat canine allergies.
The substances most likely to cause an allergic reaction vary from species to species. Common canine allergens include fleas, pollens, molds and dust. Allergy to fleas is believed to be the most common dog allergy. Typically, a flea""s saliva is the allergen, and a single fleabite can cause substantial itching. An additional form of allergy in dogs is termed atopy. Atopy is a condition where a dog is allergic to inhalants such as pollens, molds or microscopic mites such as are found in house dust.
Antibody molecules play a role in allergic manifestations. In mammals, antibody molecules are classified into various isotypes referred to as IgA, IgD, IgE, IgG, and IgM. Antibody molecules consist of both heavy and light chain components. The heavy chains of molecules of a given isotype have extensive regions of amino acid sequence homology, and conversely have regions of difference from antibodies belonging to other isotypes. The shared regions of the heavy chains provide members of each isotype with common abilities to bind to certain cell surface receptors or to other macromolecules, such as complement. These heavy chain regions, therefore, serve to activate particular immune effector functions. Accordingly, separation of antibody molecules into isotypes serves to separate the antibodies according to a set of effector functions that they commonly activate.
In humans and dogs, immunoglobulin E (hereinafter IgE) is involved in allergy. Thus, IgE is the antibody type that is understood to be an important mediator of allergic responses, including Type I immediate hypersensitivity.
IgE molecules bind to mast cells and basophils. This binding occurs when the Fc region of the IgE molecule is bound to Fc receptors on the mast cells. When such bound IgE antibodies then bind to an allergen, the allergen cross-links multiple IgE antibodies on the cell surface. This cross-linking mediates Type I immediate hypersensitivity reactions and causes release of histamines and other molecules that produce symptoms associated with allergy.
Monoclonal antibodies having different degrees of sensitivity to canine IgE and IgG have been identified. (DeBoer, et al. Immunology and Immunopathology 37, 183-199 (1993).) DeBoer, et al. identified several monoclonal antibodies which had cross reactivity between IgG and IgE. (See, e.g., DeBoer, et al., Table 4 and accompanying text.) Three monoclonal antibodies (A5, D9, and B3) were identified by DeBoer et al., as having some affinity for canine IgE. Of the monoclonal antibodies identified in DeBoer et al., antibody D9 appeared to have the greatest degree of neutralization of Prausnitz-Kustner reactivity for atopic dog serum. In the context of canine allergy, DeBoer et al. proposed use of their monoclonal antibodies (MAbs) in the use of antigen-specific IgE ELISA, and for quantifying canine IgE. Additionally, they proposed use of their MAbs for immunostaining of Western Blot assays, to evaluate the molecular specificity of IgE antibodies, as well as for in vitro studies on degranulation of mast cells.
In humans the serum level of total IgE is diagnostic of allergic disease. To explore the possibility that the serum level of IgE might also be diagnostic of allergy in dogs, several studies were performed. (Hill and DeBoer Am. J. Yet. Res., (July 1994) 55(7), 944-48). Publications following the DeBoer article used a monoclonal antibody designated D9 in an ELISA assay having the following configuration: D9 was bound to a substrate, antibodies were captured by D9 and then D9 having a marker was used to flag the captured antibody. The Hill and DeBoer ELISA was used to establish the total amount of IgE in canine serum in an effort to diagnose canine allergy. In contrast to humans, the quantity of IgE determined to exist in canine circulation was of no use whatsoever in the diagnosis of allergy in dogs. (See, e.g., Abstract and Discussion Sections of Hill and DeBoer) This finding was in direct contrast to the situation in human immunology.
This divergent diagnostic result based on levels of IgE in humans compared to such levels in dogs, points out the difficulty of any attempt to correlate data between animals of two different genera. This difficulty is further exacerbated by the fact that dogs can be allergic to a different set of antigens than humans are. Fleas, for instance, are a severe problem for dogs, but not humans. Furthermore, in instances where dogs and humans appear to be allergic to the same allergen extract, studies by doctors Esch and Greer of Greer Laboratories, have indicated that the specific allergens in an allergen extract which produce canine disease are not necessarily the same allergens that produce disease in humans. For example, it is known that the immunodominant components of dust mite extracts are different in dogs than in humans.
The genomic sequences encoding human and murine IgE heavy chain constant region are known (For example, see Ishida et al., xe2x80x9cThe Nucleotide Sequence of the Mouse Immunoglobulin E Gene: Comparison with the Human Epsilon Gene Sequencexe2x80x9d, EMBO Journal 1,1117-1123 (1982). A comparison of the human and murine genes shows that they possess 60% homology within exons, and 45-50% homology within introns, with various insertions and deletions.
Patel et al. published the nucleotide and predicted amino acid sequence for exons 1-4 of the heavy chain constant region of canine IgE in the article entitled xe2x80x9cSequence of the Dog Immunoglobulin Alpha and Epsilon Constant Region Genes,xe2x80x9d Immunogenetics 41, 282-286 (Mar. 22, 1995). The complete sequence of the canine IgE heavy chain constant region, with membrane bound portions encoded by exons 5 and 6 are disclosed in copending applications Ser. No. 08/800,698 filed Feb. 14, 1997, Ser. No. 09/146,400 filed Sep. 3, 1998, and Ser. No. 09/146,617 filed Sep. 3, 1998.
Because IgE is believed to mediate allergic symptoms, it may be desirable to decrease IgE levels as a mechanism for alleviating allergic symptoms. However, a patient""s own IgE molecules are self-proteins, and immune responses to such proteins are usually suppressed. The suppression of immune responses to self-proteins, i.e., tolerance to self-antigens, is hypothesized to occur in a number of ways.
The current hypothesis for suppression of T cells directed to self-antigens, involves an induction of xe2x80x9cclonal deletionsxe2x80x9d of such T cells in the thymus, whereby T cell receptors which might recognize self-peptides in association with MHC molecules are eliminated, and only those which recognize foreign peptide and MHC molecules are allowed to expand. In addition, suppressor T cells may also exist which prevent the induction of immune responses to self-proteins.
In contrast to the situation with T cells, it is believed that there are many B cells which express receptors (i.e., surface immunoglobulin) for self-proteins, and that the reason these cells do not produce antibodies to self-proteins is because the T cells required for the antigen presentation to the B cell are normally missing.
A B cell which recognizes epitopes (antigen-binding sites) on a patient""s own IgE antibodies is capable of generating antibodies, generally IgG, directed to this self-antigen, i.e., IgE. The existence of such B cells, therefore, presents a unique opportunity to induce the production of auto-antibody responses. There is an unmet need for such antibodies in order to treat allergic disease.
The hypothesis regarding xe2x80x9cantigen presentationxe2x80x9d involves: the recognition of antigen by surface immunoglobulin on the B cell, the internalization and processing of this antigen, the association of peptides derived from the antigen with MHC molecules expressed on the surface of the B cell, and then, the recognition of the associated antigen peptide and MHC molecules by a particular T cell. The T-cell:B-cell interaction then leads to signal transduction in both cells and the synthesis and elaboration of soluble cytokines which eventually result in antibody production by the B cell.
Thus, in most circumstances, only when an antigen is foreign does an immune response occur; otherwise the internalization and processing of self-proteins would regularly lead to the presentation of self peptide-MHC complexes to T cells and thereby lead to autoimmune antibodies.
Therefore, in order to induce an antibody response to a self-peptide, such as IgE, the immune system must be manipulated so as to allow an auto-reactive B cell to become an antibody-secreting B cell. There is an unmet need to manipulate the immune system in this way, particularly in the context of allergic disease.
In general, there are several known approaches for generating antibodies to peptide antigens. For example, multiple antigenic peptides (MAPs), introduced by Dr. James Tam (Tam, J. P., (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413), have demonstrated several advantages for inducing anti-peptide antibodies. The MAP approach is an improved alternative to the conventional technique of conjugating a peptide antigen to a protein carrier. One of the primary limitations associated with the use of protein carriers is the large mass of the carrier relative to the attached peptide antigen. This relative size disparity may result in a low ratio of anti-peptide antibodies compared to anti-carrier antibodies. MAPs typically have 4 or 8 peptide arms branching out from a lysine core matrix as depicted in FIG. 1A-B. The peptide antigen is conjugated to each arm. Thus there is a much higher ratio of antigen to carrier molecule in a MAP system compared to traditional protein conjugation. This design maximizes the concentration of the antigen for a specific immunogenic response. Moreover, the central lysine core of the MAP-peptide has been shown to be non-immunogenic. (Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413 (1988); Posnett, D. N., McGrath, H., and Tam, J. P., J. Biol. Chem. 263, 1719-1725 (1988)) Therefore, antibodies induced to MAP-peptides are a direct response to the antigen. Accurate knowledge of the chemical composition, structure, and quantity of the peptide prior to immunization is possible by directly synthesizing the antigen onto the branching lysine core. Also, because the MAP approach removes the need to conjugate peptides to carrier proteins, which may alter the antigenic determinants, chemical ambiguity is eliminated. Thus MAPs are believed to induce antibody responses of high purity, increased avidity, accurate chemical definition, and improved safety.
Fmoc MAP resins (available from Applied Biosystems, Foster City, Calif.) are Fmoc-compatible resins connected to a small core matrix of branching lysine residues. The core matrix comprises several levels of lysine residues attached to the previous lysine at both the N-xcex1 and N-xcex5 amino groups, as depicted in FIGS. 1A-B.
MAP-peptides used in experimental vaccine design have elicited high titers of anti-peptide antibodies that recognize the native protein. (Tam, J. P., (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413; Posnett, D. N., McGrath, H., and Tam, J. P., (1988) J. Biol. Chem. 263, 1719-1725; Auriault, C., Wolowczuk, I., Gras-Masse, H., Maguerite, M., Boulanger, D., Capron, A., and Tartar, A., (1991) Peptide Res.4, 6-11). Additionally, increased sensitivity and reliability of antibody-antigen interactions in solid-phase immunoassays have been observed with MAP-peptides due to enhanced coating capacity and avidity. (Tam, J. P., and Zavala, F., (1989) J. Immunol. Meth., 124, 53-61).
An additional approach that is known to be useful for generating antibodies to peptide antigens involves placing multiple copies of peptides on the surface of plant virus particles. EPICOAT(trademark) technology (Axis Genetics plc, Cambridge, England) is one such example. The EPICOAT(trademark) technology is based on chimeric virus particle (CVP) technology that utilizes the recombinant genetic modification of plant viruses.
The EPICOAT(trademark) technology involves insertion of a small portion of a foreign protein (a peptide) into a plant virus in such a way that multiple copies of the peptide are displayed on the surface of the virus particle. The EPICOAT(trademark) technology is currently based on the cow pea mosaic virus (CPMV) a plant virus that infects the cow pea plant, also known as the xe2x80x9cblack-eyedxe2x80x9d bean. The unmodified CPMV particle is icosahedral, and about twenty-eight nanometers (nm.) in diameter. CPMV particles are composed of two proteins, referred to as the large and small coat proteins. Studies have revealed a site within the small coat protein which allows presentation of a foreign peptide in a prominent position on the virus surface, whereby up to sixty copies of a particular peptide can be presented on each virus particle.
With the EPICOAT(trademark) technology, DNA copies of the plant virus""s genetic material are used. A minute quantity of the DNA encoding the virus protein, including the inserted foreign peptide, is applied to the leaves of young cow pea plant, along with an abrasive powder. Upon gentle rubbing, DNA enters the leaves and utilizes the plant""s own cellular mechanisms to initiate generation of functional virus particles. The virus replicates within the inoculated leaves and spreads throughout the growing plant.
After two to three weeks, leaf material containing large quantities of the virus is harvested. Chimeric virus particles (CVPs) are isolated by centrifugation and selective precipitation of homogenized plant material. Between 1 to 2 grams of CVPs can be obtained per kilogram fresh weight of leaf material. Cow pea plants are readily grown in abundance in controlled environments, allowing generation of large quantities of CVPs.
It has been reported that peptides of up to thirty-six amino acids in size have been successfully incorporated into CVPs. The resulting particles are extremely robust with a thermal inactivation point of 65xc2x0 C. The CVPs have been shown to withstand acidic pH as well as protein degrading enzymes. It has been reported that the expressed peptides are capable of eliciting specific immune responses in animals. It has been hypothesized that surface presentation of peptides may enhance the recognition by the host immune system, and may provide a route for development of recombinant sub-unit vaccines and immuno-therapeutics.
Disclosed is a specific binding protein which specifically binds to native canine free or B cell-bound IgE exon 3, and which does not bind to IgE exon 3 when the IgE is bound to receptor on a mast cell. The surface-bound IgE can be IgE expressed on the surface of a canine B cell.
Disclosed is a specific binding protein which specifically binds to an isolated and purified peptide comprising a leucine positioned two peptide bonds away from a tyrosine-arginine pair; e.g., SEQ ID NO:1 leucine-blank-blank-tyrosine-arginine, SEQ ID NO:2 tyrosine-arginine-blank-blank-leucine, or SEQ ID NO:3 leucine-blank-blank-tyrosine-arginine-blank-blank-leucine. The peptide can consist of from 5 to 71 amino acids.
Disclosed is an antibody which binds to a defined epitope and which is raised to an isolated and purified peptide comprising an amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO:4 Thr-Leu-Leu-Glu-Tyr-Arg-Met; also disclosed is a recombinant binding molecule which specifically binds to the defined epitope bound by the antibody.
Disclosed is an antibody which binds to a defined epitope and which is raised to an isolated and purified peptide comprising an amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO:5 Gly-Met-Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys; also disclosed ii a recombinant binding molecule which specifically binds to the defined epitope bound by the antibody.
Also disclosed is a specific binding protein which is raised to a multiply antigenic peptide comprising multiple copies of an isolated and purified peptide which comprises a leucine positioned two peptide bonds away from a tyrosine-arginine pair; the specific binding protein specifically binds to a defined epitope. The isolated and purified peptide can be from 5-71 amino acids. Also disclosed is a recombinant binding molecule which specifically binds to the defined epitope bound by the binding protein.
Disclosed is a specific binding protein which specifically binds to an isolated and purified peptide comprising a cysteine positioned two peptide bonds away from a proline-histidine pair positioned three peptide bonds from a cysteine. The peptide can have the form: SEQ ID NO:6 cysteine-blank-blank-proline-histidine-blank-blank-blank-cysteine. The cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine. The peptide can comprise from 9 to 71 amino acids.
Disclosed is an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO:7 serine-valine-threonine-leucine-cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine-glycine-glycine-glycine. The cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine. Also disclosed is a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
Disclosed is an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO:8 serine-alanine-cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine-glycine-glycine-glycine. The cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide changing the amino acid to cystine. Also disclosed is a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
Disclosed is a specific binding protein that specifically binds to an isolated and purified peptide comprising a cysteine positioned one peptide bond from a proline-histidine pair, positioned one peptide bond from a proline, positioned two peptide bonds from a cysteine; the peptide can have the form: SEQ ID NO:9 cysteine-blank-proline-histidine-blank-proline-blank-blank-cysteine. The cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine. The peptide can comprise from 9 to 71 amino acids.
Disclosed is an antibody that binds to a defined epitope and which is raised to an isolated and purified peptide that has the amino acid sequence, or a conservative variant thereof, which comprises: SEQ ID NO:10 serine-alanine-cysteine-histidine-proline-histidine-leucine-proline-lysine-serine-cysteine-glycine-glycine-glycine. The cysteines may form a covalent bond through a reduction reaction, cyclizing the peptide and becoming cystine. Also disclosed is a recombinant binding molecule that specifically binds to the defined epitope bound by the antibody.
Specific binding proteins in accordance with the invention can be antibodies, either polyclonal or monoclonal. For example the monoclonal antibody can be 8H.8 or 15A.2. The antibody in accordance with the invention will bind a defined epitope; also disclosed are recombinant binding molecules which specifically binds to the defined epitope bound by an antibody in accordance with the invention.
Also disclosed is a method for treatment or prophylaxis for canine allergy, said method comprising: providing a specific binding molecule in accordance with the invention; and, administering the provided binding molecule to a dog. The method can comprise a step of mixing the provided binding molecule with a diluent prior to the administering step, and wherein the administering step comprises administering the mixture of the molecule and the diluent. A step of administering a booster dose of the binding molecule can follow the administering step.
cDNA clone: A duplex DNA sequence representing an RNA, carried in a cloning vector.
Cloning: The selection and propagation of a single DNA species.
Cloning Vector: A plasmid, phage DNA or other DNA sequence, able to replicate in a host cell and capable of carrying exogenously added DNA sequence for purposes of amplification or expression of the added DNA sequence.
Codon: A triplet of nucleotides that represents an amino acid or termination signal.
Conservative variants: Conservative variants of nucleotide sequences include nucleotide substitutions that do not result in changes in the amino acid sequence encoded by such nucleotides, as well as nucleotide substitutions that result in conservative amino acid substitutions, e.g., amino acid substitutions which do not substantially affect the character of the polypeptide translated from said nucleotides. For example, the character of a peptide derived from IgE is not substantially affected if the substitutions do not preclude specific binding of the peptide to canine IgE receptor or other canine IgE binding ligands.
Conservative variants of amino acid sequences include amino acid substitutions or deletions that do not substantially affect the character of the variant polypeptide relative to the starting peptide. For example, polypeptide character is not substantially affected if the substitutions or deletions do not preclude specific binding of the variant peptide to a specific binding partner of the starting peptide. The term mimotope refers to a conservative variant of an amino acid sequence, to which antibody specificity has been raised. The mimotope comprises a variant of the epitope of the starting peptide such that it is able to bind antibodies that cross-react with the original epitope.
DNA Sequence: A linear series of nucleotides connected one to the other by phosphodiester bonds between the 3xe2x80x2 and 5xe2x80x2 carbons of adjacent pentoses.
Expression: The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Expression Control Sequence: A DNA sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes.
Exon: A contiguous region of DNA encoding a portion of a polypeptide. Reference to any exon, e.g. xe2x80x9cDNA sequence of exon 6xe2x80x9d, refers to the complete exon or any portion thereof.
Genome: The entire DNA of a substance. It includes inter alia the structural genes encoding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interaction sequences such as the Shine-Dalgarno sequences.
Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The four DNA bases are adenine (xe2x80x9cAxe2x80x9d), guanine (xe2x80x9cGxe2x80x9d), cytosine (xe2x80x9cCxe2x80x9d) and thymine (xe2x80x9cTxe2x80x9d). The four RNA bases are A, G, C and uracil (xe2x80x9cUxe2x80x9d). A and G are purines, and C, T, and U are pyrimidines.
Phage or Bacteriophage: Bacterial virus, many of which include DNA sequences encapsidated in a protein envelope or coat (xe2x80x9ccapsidxe2x80x9d).
Plasmid: An autonomous self-replicating extrachromosomal circular DNA.
Polymerase Chain Reaction (PCR): A method of amplifying a target DNA sequence contained in a mixture of DNA sequences, by using oligonucleotide primers that flank the target DNA sequence for repeated cycles of DNA synthesis of the target DNA sequence.
Polypeptide: A linear series of amino acids connected one to the other by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
Reading Frame: The grouping of codons during translation of mRNA into amino acid sequences. For example, the sequence GCTGGTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GOT TGT AAG-Ala-Gly-Cys-Lys
G CTG GTT GTA AG-Leu-Val-Val
GC TGG TTG TAA A-Trp-Leu-(STOP).
Recombinant DNA Molecule: A hybrid DNA sequence comprising at least two nucleotide sequences, the first sequence not normally being found together in nature with the second.
Specific binding: Binding of one substance to another at greater binding affinity than background binding. Two substances that exhibit specific binding are referred to as specific binding partners, or as a specific binding pair. An antibody and its antigen are one example of a specific binding pair.
Specific Binding Molecule: A molecule that exhibits specific binding to its corresponding binding partner to form a specific binding pair. As used herein, this definition of specific binding molecule covers monoclonal and polyclonal antibodies, antigen-binding fragments of these antibodies, hybrid antibodies, single-chain antibodies, and recombinant molecules capable of specific binding to a ligand.
Structural Gene: A DNA sequence that encodes through its template or messenger RNA (xe2x80x9cmRNAII) a sequence of amino acids characteristic of a specific polypeptide.
Transcription: Synthesis of RNA on a DNA template.
Translation: Synthesis of peptides on the mRNA template.